Metagenomics of Otitis Media
Otitis Media (OM) is the inflammation of the middle ear, from the tympanic membrane to the colchea, including the eustacian tube). It is highly prevalent worldwide, and is commonly found in children. 83% of all children will have at least one experience of acute OM (AOM) by age 3 and 40% of children will experience more than 3 episodes of AOM. This is a major healthcare concern worldwide, especially to children due to the immaturity of the pediatric immune system, along with many other factors such as the length of the eustachian tube which is significantly shorter in children, allowing easier entry of microorganisms into the middle ear. OM is a sequelae of upper respiratory tract infections and can be caused by combinatory effects of viral, bacterial, and fungal pathogens. Commonly found in OM are S. pneumoniae, Haemophilius influenzae, and Moraxella catarrhalis, which cause OM 85% of the time. The remaining 15% of cases are caused by viral infections. The most common treatment for OM is the antibiotic amoxicillin. The two microbes that the following paper focuses on are H. influenzae and M. catarrhalis. H. influenzae is a gram-negative coccobaccillary facultative anaerobe (3) and M. catarrhalis is a fastidious, non-motile, gram-negative, aerobic diplococcus known to cause human infections of the respiratory system, middle ear, eye, CNS, and joints (4) Indirect Pathogenicity of Haemophilus inﬂuenzae and Moraxellacatarrhalis in Polymicrobial Otitis Media Occurs via InterspeciesQuorum Signaling (2) Background This paper discusses how the polymicrobial nature of OM affects its course, severity, and treatability. They primarily focused on the presence of H. influenzae and M. catarrhalis and their formation of biofilms, promoting resistance from immune clearance and also antibiotic administration. This study shows how communication between bacterial species promotes antibiotic resistance and persistance in OM. Results H. influenzae and M. catarrhalis form polymicrobial biofilms in vitro Static biofilms of H. influenzae, M. catarrhalis, or both were established in miscroscopy chamber slides, and the surface-attached microbial communites were examined using SEM (Figure 1A) and CLSM (Figure 1B) at various time points during biofilm development. It was found that H. influenzae formed matrix-encased biofilm communities and M. catarrhalis formed smaller surface-attached clusters. When co-cultured, both species were encorporated within polymicrobial biofilms, M. catarrhalis being larger diplococci interspersed with smaller H. influenzae coccobacilli (Figure 1A). M. catarrhalis communities were located in discrete regions among the H. influenzae biofilm structure, seen in figure 1B. Polymicrobial Biofilms Provide Passive Antibiotic Resistance Beta-lactam antibiotics are used for the treatment of OM commonly and it M. catarrhalis strains are virtually universally resistant to Beta-lactam drugs via the secretion of beta-lactamases (inhibition of beta lactam antibiotics). It has also been hypothesized that passive resistance could be achieved during coinfection with M. catarrhalis as a mechanism used by many airway pathogens. To see if M. catarrhalis could provide passive protection for H. influenzae (beta-lactam sensitive) within a polymicrobial biofilm, biofilms were established for 24 hours and were treated with ampicillin (a beta-lactam antibiotic) and the beta-lactamase inhibitor clavulanate, shown in figure 2. Results showed that biofilms formed by H. influenzae were susceptible to the ampicillin treatment while M. catarrhalis biofilms were resistant. Polymicrobial biofilms resulted in increased recovery of H. influenzae, bringing them to the conclusion that M. catarrhalis provides protection against ampicillin treatment. However, when clavulanate was added, the recovery of H. influenzae was back to the level seen when M. catarrhalis was absent, leading to the conclusion that the increased protection was in fact due to the beta-lactamase produced by the M. catarrhalis. As a control, clavulunate was seen to also reduce the recovery of M. catarrhalis colonies from single-species biofilms. Another interesting result was that when the two species were co-cultured, there was also an increase in the recovery of viable M. catarrhalis as well as H. influenzae, even when clavulanate was present, due to the properties of polymicrobial biofilms, which increase protection of micrbial colonies. Next, they used trimethoprim-sulfamethoxazole to determine if polymicrobial biofilms provide passive protection that is independent of diffusible resistance determinants. They found that H. influenzae biofilms were less resistant to this combination of antibiotics whereas M. catarrhalis biofilms were more resistant. Polymicrobial biofilms formed by the combination illustrated protection to H. influenzae from trimethoprim-sulfamethoxazole (Figure 3A). This result shows that antibiotic protection can be provided by the polymicrobial biofilm independently of genetic changes or transfer of resistance determinants between species in co-culture. To determine the protective capabilities of H. influenzae biofilm against antibiotics within a polymicrobial biofilm, clarithromycin (a macrolide to which M. catarrhalis is susceptible) was used at a concentration that eradicates M. catarrhalis. it was shown that polymicrobial biofilms formed by the combination protected M. catarrhalis from the lethal-concentration of clarithromycin, shown by a significant increase in recovered M. catarrhalis bacteria. It is important to note that amount of protection provided to M. catarrhalis by H. influenzae when present in polymicrobial biofilms together is dependent on if H. influenzae contained a biofilm defect and if so, which H. influenzae biofilm defects were present, either siaB, licD, or lux S (Figure 3B). It was seen that H. influenzae mutants with biofilm defects siaB, licD, and luxS had significantly diminished protection in M. catarrhalis, when compared to that of the parental strain. It was also seen that protection of M. catarrhalis was increased in biofilms formed with H. influenzae licON, a mutant forming thicker biofilms, and thus offering greater protection against antibiotics. To ensure that increased antibiotic resistance was not due to genetic changes or transfer of resistant determinants between species, control experiments showed that bacteria recovered from all polymicrobial biofilms retained susceptibility characteristics similiar to those of the inocula. Autoinducer-2 Promotes M. catarrhalis biofilm thickness and antibiotic resistance M. catarrhalis is not known to possess a luxS homolog and did not produce AI-2 during growth in broth culture, but recent evidence indicates that bacterial species which do not make AI-2 may still sense and respond to the AI-2 signal. To test this, M. catarrhalis was cultured in broth supplemented with chemically-synthesized AI-2 precursor DPD, and samples were taken to measure the amount of DPD remaining in the culture over time (figure 4A). M. catarrhalis depleted DPD in 6h, indicating the uptake or degradation of DPD. However, uninoculated controls showed a minimal decrase in the AI-2 signal after 6h. Also interesting, the amount of DPD depleted by M. catarrhalis was simliar to the amount depleted by H. influenzae luxS. To determine if AI-2 requires live bacteria or protein synthesis, M. catarrhalis cultures were incubated with tetracycline overnight prior to DPD addition or were incubated with tetracycline concurrently with DPD. samples were taken over the course of 7h for comparison of M. catarrhalis DPD depletion to that of untreated M. catarrhalis (figure 4B). Both tetracycline treatments completely inhibited depletion of DPD by M. catarrhalis, suggesting that depletion is an active process requiring protein synthesis. Thus the conclusion was made that M. catarrhalis is most likely depleting AI-2 by means of an uptake system instead of external degradation. To determine the result of AI-2 on M. catarrhalis biofilm formation, M. catarrhalis biofilms were established in the presence or absense of DPD and stained with crystal violet at 4, 6, 12, 24, and 48h (fig 4C). Presence of DPD resulted in an increase in M. catarrhalis biofilm biomass , especially at 24 and 48h (this increase was confirmed by viability staining and CLSM). In the absences of DPD, M. catarrhalis biofilms formed small clusters with mostly nonviable staining (figure 4D), and those with DPD present formed thicker biofilms and resulted in an increased number of viable bacteria within the larger biomasses (fig. 4E). From the results, it was hypothesized that DPD increases resistance of M. catarrhalis to antibiotic treatment. To specifically test this hypothesis, M. catarrhalis biofilms were established in the presence or absence of DPD and were then treated with clarithromycin. It was seen that DPD treatment did not significantly alter the recovery of viable M. catarrhalis from control wells. However, M. catarrhalis biofilms established in the presence of DPD were significantly more resistant to the antibiotic, indicated by an increased recovery of viable M. catarrhalis bacteria following incubation with the drug. Conclusions were made stating that M. catarrhalis does not produce AI-2, this strain does respond to the interspecies quorum signaling molecule DPD by producing biofilms with increased biomass and resistance to antibiotics. Interspecies Quorum signaling during polymicrobial infection promotes persistance of M. catarrhalis Because M. catarrhalis is commonly isolated from polymicrobial OM infections, it was hypothesized that the bacteria could utilize AI-2 produced by H. influenzae or other OM pathogens to persist in vivo. To test this directly, the chinchilla model of OM was utilized. It was found that M. catarrhalis survives exclusively in surface-attached communities within the chinchilla middle ear chamber and that coinfection with H. influenzae provides a permissive environment in which M. catarrhalis can proliferate. Next, they wondered if the increase in numbers of M. catarrhalis during coinfection with H. influenzae was dependent on interspecies quorum signaling. Based, on the results obtained here and previously stated, it was concluded that M. catarrhalis can utilize exogenous AI-2 provided by H. influenzae to establish a persistant infection. Conclusion This metagenomic analysis illustrates indirect pathogenicity and that bacterial disease and/or treatment response is dependent on the influences of other bacteria within the same environment. H. influenzae protects M. catarrhalis and promotes persistance and antibiotic resistance via response to interspecies quorum signaling. M. catarrhalis was also illustrated to act on H. influenzae as an indirect pathogen through the production of beta-lactamase. This study provides evidence for polymicrobial infection influence on the severity of disease and also the effectiveness of antibiotic treatment, especially for chronic infections involving persistance of bacteria within biofilms. This indicates that vaccination against upper airway pathogens, like H. influenzae, could actually impact both H. influenzae infection but also M. catarrhalis infections. References 1. http://www.medicinenet.com/ear_infection/article.htm (Fig.1) 2. http://hmjournalclub.files.wordpress.com/2011/06/mbio-2010-armbruster.pdf 3. http://en.wikipedia.org/wiki/Haemophilus_influenzae 4. http://en.wikipedia.org/wiki/Moraxella_catarrhalis